Combined therapeutic effect of YHO-1701 with PD-1 blockade is dependent on natural killer cell activity in syngeneic mouse models.

The signal transducer and activator of transcription 3 (STAT3) signaling pathway is a key mediator of cancer cell proliferation, survival, and invasion. We discovered YHO-1701 as a small molecule inhibitor of STAT3 dimerization and demonstrated its potent anti-tumor activity using xenograft mouse models as monotherapy and combination therapy with molecular targeted drugs. STAT3 is also associated with cancer immune tolerance; therefore, we used the female CT26 syngeneic mouse model to examine the effect of combining YHO-1701 administration with PD-1/PD-L1 blockade. Pretreatment of the mice with YHO-1701 before starting anti-PD-1 antibody administration resulted in a significant therapeutic effect. In addition, the effect of monotherapy and combination treatment with YHO-1701 was significantly abolished by depleting natural killer (NK) cell activity. YHO-1701 was also found to restore the activity of mouse NK cells under inhibitory conditions in vitro. Furthermore, this combination therapy significantly inhibited tumor growth in an immunotherapy-resistant model of murine CMS5a fibrosarcoma. These results suggest that the combination of YHO-1701 with PD-1/PD-L1 blockade might be a new candidate for cancer immunotherapy involving the enhancement of NK cell activity in the tumor microenvironment.

Design, synthesis, and evaluation of a novel prodrug, a S-trityl-l-cysteine derivative targeting kinesin spindle protein.

Kinesin spindle protein (KSP) is expressed only in cells undergoing cell division, and hence represents an attractive target for the treatment of cancer. Several KSP inhibitors have been developed and undergone clinical trial, but their clinical use is limited by their toxicity to rapidly proliferating non-cancerous cells. To create new KSP inhibitors that are highly selective for cancer cells, we optimized the amino acid moiety of S-trityl-l-cysteine (STLC) derivative 1 using in silico modeling. Molecular docking and molecular dynamics simulation were performed to investigate the binding mode of 1 with KSP. Consistent with the structure activity relationship studies, we found that a cysteine amino moiety plays an important role in stabilizing the interaction. Based on these findings and the structure of GSH, a substrate of γ-glutamyltransferase (GGT), we designed and synthesized the prodrug N-γ-glutamylated STLC derivative 9, which could be hydrolyzed by GGT to produce 1. The KSP ATPase inhibitory activity of 9 was lower than that of 1, and LC-MS analysis indicated that 9 was converted to 1 only in the presence of GGT in vitro. In addition, the cytotoxic activity of 9 was significantly attenuated in GGT-knockdown A549 cells. Since GGT is overexpressed on the cell membrane of various cancer cells, these results suggest that compound 9 could be a promising prodrug that selectively inhibits the proliferation of GGT-expressing cancer cells.

Discovery of a Potent Anticancer Agent PVHD303 with in Vivo Activity.

As a part of our continuous structure-activity relationship (SAR) studies on 1-(quinazolin-4-yl)-1-(4-methoxyphenyl)ethan-1-ols, the synthesis of derivatives and their cytotoxicity against the human lung cancer cell line A549 were explored. This led to the discovery of 1-(2-(furan-3-yl)quinazolin-4-yl)-1-(4-methoxyphenyl)ethan-1-ol (PVHD303) with potent antiproliferative activity. PVHD303 disturbed microtubule formation at the centrosomes and inhibited the growth of tumors dose-dependently in the HCT116 human colon cancer xenograft model in vivo.

Discovery of a New STAT3 Inhibitor Acting on the Linker Domain.

Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor that contributes to tumor cell growth and survival and is often constitutively active in several types of cancers, which makes it an attractive target for cancer therapy. We identified 5,5'-(pentane-1,5'-diyl)bis(2-methyl-1,4-benzoquinone) (BPMB) as a new STAT3 inhibitor. BPMB inhibited the transcriptional activities of STAT3, despite its inability to reduce the phosphorylation and nuclear translocation of STAT3. BPMB selectively inhibited the proliferation of human breast cancer cell lines with constitutively activated STAT3. Furthermore, a gel retardation pattern was obtained by immunoblotting only when those STAT3-activated cell lines were treated with BPMB. The shifted bands could be immunoblotted with anti-STAT3 antibody but not with anti-STAT1/STAT5 antibody, and were stable under reducing conditions. The purified recombinant STAT3 protein treated with BPMB afforded a similar band shift pattern. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of the component comprising the main shifted band suggested that the complex is a STAT3 homodimer crosslinked by BPMB through a Michael addition with Cys550 in the linker domain. Alanine replacement at this position resulted in reduction of the STAT3 dimer formation in the gel retardation assay. Thus, our results suggest that BPMB inhibits the proliferation of STAT3-activated cell lines, presumably through acylation of the linker domain and subsequent induction of the inactive STAT3 complexes.

An in vivo active 1,2,5-oxadiazole Pt(II) complex: A promising anticancer agent endowed with STAT3 inhibitory properties.

New Pt(II) complexes (Pt-1-3) bearing 1,2,5-oxadiazole ligands (1-3) were synthesized, characterized and evaluated for their ability to disrupt STAT3 dimerization. Ligand 3·HCl showed cytotoxic effects on HCT-116 cells (IC50 = 95.2 µM) and a selective ability to interact with STAT3 (IC50 = 8.2 µM) versus STAT1 (IC50 > 30 µM). Its corresponding platinum complex Pt-3 exhibited an increased cytotoxicity (IC50 = 18.4 µM) and a stronger interaction with STAT3 (IC50 = 1.4 µM), leading to inhibition of its signaling pathway. Pt-3 was also evaluated in cell-based assays for its action on p53 expression and on STAT3 phosphorylation. In syngeneic murine Lewis lung carcinoma (LLC) implanted in C57BL/6 mice, Pt-3 showed a higher antitumor activity with fewer side effects than cisplatin.

Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity.

Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors.

Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins.

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.